Hla homozygous cells and methods of use thereof

ABSTRACT

The present invention provides methods of generating a mammalian cell that is homozygous at a locus of interest, as well as cells made by the method. The present invention further provides methods of using the cells.

CROSS-REFERENCE

This application is a Continuation application of U.S. patentapplication Ser. No. 12/044,471 filed Mar. 7, 2008, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/905,966, filed Mar. 9, 2007, the content of which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. government may have certain rights in this invention, pursuantto grant nos. P20 GM69983 and R01 DK55759 awarded by the NationalInstitutes of Health.

BACKGROUND

Transplantation of functional cells and tissues from a donor individualinto a recipient is a major means of replacing failing or non-functionalorgans or tissues in the recipient. One of the major obstacles tosuccessful transplantation is rejection of the transplanted cells ortissues by the recipient's immune system, due to tissue incompatibility.Immunosuppressive drugs such as cyclosporin A are currently used toreduce transplant rejection. However, immunosuppressive drugs arecostly, toxic, and sometimes ineffectual. In humans, molecules encodedby the human leukocyte antigen (HLA) locus provide the context for therecognition of “non-self” or foreign cells by the transplant recipient.

The histocompatibility type of an individual is determined by many geneslocated in the HLA locus on human chromosome 6. Every person has twocopies of the HLA locus—one on each copy of chromosome 6. There areseveral different versions or “haplotypes” of the genes in the HLAlocus, and since there are two copies of each gene, it is very unlikelythat two individuals have the same versions of each gene or “HLA type.”

To ensure that transplants, such as bone marrow transplants, are notrejected by a recipient, attempts are made to use a donor with the sameHLA type. The chances that a prospective recipient's sibling will havethe same HLA type as the recipient are about 1 in 4. Therefore, siblingsare usually used as donors when performing bone marrow transplantation.If no HLA-matched sibling is available, then an unrelated, HLA-matched,donor can sometimes be found; however, this requires searching throughthousands of potential donors in the unrelated bone marrow donorregistry to find an HLA match. More typically, where an unrelated donoris used, an exact match is not found; instead, a near-match is used.

In solid organ transplantation, HLA matching is typically not possible,due to the limited number of donors. Because organ transplantationtypically involved HLA unmatched donors and recipients, aggressiveimmunosuppressive medications are typically required. These medicationsare very toxic and often fail to prevent transplant rejection.

There is an ongoing need in the art for HLA matched stem cells that canserve as donors in various therapeutic transplant settings.

LITERATURE

U.S. Pat. No. 7,030,292; WO 98/48005; U.S. Pat. No. 6,986,887.

SUMMARY OF THE INVENTION

The present invention provides methods of generating a mammalian cellthat is homozygous at a locus of interest, as well as cells made by themethod. The present invention further provides methods of using thecells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the AAV2-HMGA-1-HyTK vector.

FIG. 2 is a schematic depiction of selection for homozygosity at the HLAlocus.

FIG. 3 depicts Class I HLA alleles.

FIG. 4 depicts Class II HLA alleles.

FIG. 5 is a schematic depiction of human HLA genes.

FIG. 6 is a graph depicting HLA-A,B coverage in U.S. populations.

FIG. 7 depicts common HLA-A,B haplotypes in five U.S. populations.

DEFINITIONS

As used herein, the term “stem cell” refers to an undifferentiated cellthat can be induced to proliferate. The stem cell is capable ofself-maintenance or self-renewal, meaning that with each cell division,one daughter cell will also be a stem cell. Stem cells can be obtainedfrom embryonic, post-natal, juvenile, or adult tissue. Stem cells can bepluripotent or multipotent. The term “progenitor cell,” as used herein,refers to an undifferentiated cell derived from a stem cell, and is notitself a stem cell. Some progenitor cells can produce progeny that arecapable of differentiating into more than one cell type.

As used herein, the term “genetic modification” refers to a permanent ortransient genetic change induced in the genome of a cell followingintroduction of new nucleic acid (i.e., DNA exogenous to the cell).Genetic modification can be accomplished by incorporation of the new(“exogenous”) DNA into the genome of the host cell, or by transient orstable maintenance of the new DNA as an episomal element. Geneticmodifications include, e.g., duplication of an endogenous nucleotidesequence in the nuclear genome. Following induction of a genetic changein the genome (e.g., the nuclear genome) of a cell, the exogenous DNAcan remain in the cell (e.g., can be integrated into the genome, or canbe present extrachromosomally), or can be absent from the cell (e.g.,deleted from the genome). Means for effecting a “genetic modification”exclude parthenogenesis.

A “parvoviral vector” refers to a vector based on or derived from aparvovirus such as adeno-associated virus (AAV) and minute virus of mice(MVM). See, e.g., Hendrie et al. (2003) J. Virol. 77:13136-13145; andRussell et al. (2002) Nat. Biotech. 20:658.

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”). The term “AAV” includes, but is not limited to, AAV type1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4),AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type8 (AAV-8).

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two AAV inverted terminalrepeat sequences (LTRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Packaging” refers to a series of intracellular events that result inthe assembly and encapsidation of an AAV particle.

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans.

An “infectious” virus or viral particle is one that comprises apolynucleotide component which it is capable of delivering into a cellfor which the viral species is trophic. The term does not necessarilyimply any replication capacity of the virus. Assays for countinginfectious viral particles are described elsewhere in this disclosureand in the art. Viral infectivity can be expressed as the P:I ratio, orthe ratio of total viral particles to infective viral particles.

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound or a number of cells that, when administered to amammal or other subject for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on one or more factors such as the cell, the diseaseand its severity and the age, weight, etc., of the subject to betreated.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anHLA homozygous cell” includes a plurality of such cells and reference to“the rAAV vector” includes reference to one or more rAAV vectors andequivalents thereof known to those skilled in the art, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides cells that are genetically modified suchthat they are homozygous at a human leukocyte antigen (HLA) locus. TheseHLA homozygous cells are compatible with any recipient containing asingle copy of that HLA locus present in the HLA homozygous cells,regardless of the other haplotype present in the recipient. Thus, onlyone HLA haplotype need be matched. The HLA homozygous cells can also beintroduced into individuals who have an HLA haplotype that is not anexact match of the HLA haplotype in the HLA homozygous cell.

An HLA haplotype is a collection of HLA class I and class II allelesfound on a given chromosome in an individual. Currently known HLA classI and class II alleles are depicted in FIGS. 3 and 4, respectively. Thehuman HLA locus is depicted schematically in FIG. 5. HLA-A,B haplotypecoverage in U.S. populations is depicted in FIG. 6; and common HLA-A,Bhaplotypes in five U.S. populations are depicted in FIG. 7. For FIG. 7,see Faden et al. (2003) Hastings Center Report 33:13.

The present invention provides a repository (e.g., a library) of HLAhomozygous cells. The HLA haplotypes represented in a subject librarycan reflect the most common HLA haplotypes found in human populations,e.g., common Caucasian HLA haplotypes, common HLA haplotypes found inindividuals of African ancestry, common Asian HLA haplotypes, commonHispanic HLA haplotypes, common Native American HLA haplotypes, etc. Forexample, a single abundant haplotype can be present in a significantproportion of a population, allowing a single HLA homozygous cell lineto serve as a histocompatible donor for a significant percent ofpatients. The library of cells can include HLA homozygous cells of thesame type; HLA homozygous stem cells of the same cell type; HLAhomozygous cells of two or more different cell types; etc.

The HLA homozygous cells described herein can find use in a broad arrayof clinical applications involving transplantation of cells and/ortissues. The HLA homozygous cells are HLA compatible with a recipient,and therefore can be introduced into the recipient without the need forimmunosuppressive therapy, or at least with reduced need forimmunosuppressive therapy. A standard immunosuppressive drug regimencosts thousands of dollars per month, and can have undesirable sideeffects, including infections and cancers that are oftenlife-threatening and expensive to treat. The present HLA homozygouscells thus overcome some of the obstacles currently limiting the use ofhuman cells for clinical applications.

Genetically Modified, HLA Homozygous Cells

The present invention provides genetically modified, HLA homozygoushuman cells. In some cases, the HLA homozygous cell is an HLA homozygousstem cell. In other cases, the HLA homozygous cell is a progenitor cell.In still other cases, the HLA homozygous cell is a terminallydifferentiated cell. The present invention also provides progeny of asubject HLA homozygous stem cell, including progeny that aredifferentiated. Thus, e.g., the present invention provides HLAhomozygous cardiomyocytes; HLA homozygous differentiated progeny of ahematopoietic stem cell; HLA homozygous cartilage cells; HLA homozygouspancreatic islet cells; and the like.

Subject HLA homozygous cells are generated by genetic modification of anHLA heterozygous parent stem cell, using methods that are described inmore detail below. Features of the parent HLA heterozygous cells aredescribed below.

Parent HLA Heterozygous Cells

HLA heterozygous cells that are genetically modified to be HLAhomozygous are referred to herein as “parent HLA heterozygous cells” orsimply “parent cells.” Parent cells that are can be genetically modifiedto generate a subject HLA homozygous cell include, but are not limitedto, stem cells, e.g., hematopoietic stem cells, embryonic stem cells,mesenchymal stem cells, neural stem cells, epidermal stem cells,endothelial stem cells, gastrointestinal stem cells, liver stem cells,cord blood stem cells, amniotic fluid stem cells, pancreatic stem cells,and the like; as well as differentiated cells that can be cultured invitro and used in a therapeutic regimen, where such cells include, butare not limited to, keratinocytes, adipocytes, cardiomyocytes,pancreatic islet cells, retinal cells, and the like. The parent cellthat is used will depend in part on the nature of the disorder orcondition to be treated.

Suitable parent human embryonic stem (ES) cells include, but are notlimited to, any of a variety of available human ES lines, e.g.,BG01(hESBGN-01), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen, Inc.;Athens, Ga.); SA01 (Sahlgrenska 1), SA02 (Sahlgrenska 2) (Cellartis AB;Goeteborg, Sweden); ES01 (HES-1), ES01 (HES-2), ES03 (HES-3), ES04(HES-4), ES05 (HES-5), ES06 (HES-6) (ES Cell International; Singapore);UC01 (HSF-1), UC06 (HSF-6) (University of California, San Francisco; SanFrancisco, Calif.); WA01 (H1), WA07 (H7), WA09 (H9), WA13 (H13), WA14(H14) (Wisconsin Alumni Research Foundation; WARF; Madison, Wis.). Cellline designations are given as the National Institutes of Health (NIH)code, followed in parentheses by the provider code. See, e.g., U.S. Pat.No. 6,875,607.

Methods of culturing human ES cells are known in the art. See, e.g.,U.S. Pat. No. 6,875,607. Human ES cells can be cultured in vitro usingany known method.

Suitable parent human ES cell lines can be positive for one, two, three,four, five, six, or all seven of the following markers: stage-specificembryonic antigen-3 (SSEA-3); SSEA-4; TRA 1-60; TRA 1-81; Oct-4; GCTM-2;and alkaline phosphatase. Parent human ES cell lines can be negative forSSEA-1.

Hematopoietic stem cells (HSCs) are mesoderm-derived cells that can beisolated from bone marrow, blood, cord blood, fetal liver and yolk sac.HSCs are characterized as CD34⁺ and CD3⁻. HSCs can repopulate theerythroid, neutrophil-macrophage, megakaryocyte and lymphoidhematopoietic cell lineages in vivo. In vitro, HSCs can be induced toundergo at least some self-renewing cell divisions and can be induced todifferentiate to the same lineages as is seen in vivo. As such, HSCs canbe induced to differentiate into one or more of erythroid cells,megakaryocytes, neutrophils, macrophages, and lymphoid cells.

Neural stem cells (NSCs) are capable of differentiating into neurons,and glia (including oligodendrocytes, and astrocytes). A neural stemcell is a multipotent stem cell which is capable of multiple divisions,and under specific conditions can produce daughter cells which areneural stem cells, or neural progenitor cells that can be neuroblasts orglioblasts, e.g., cells committed to become one or more types of neuronsand glial cells respectively. Methods of obtaining and culturing NSCsare known in the art.

Mesenchymal stem cells (MSC), originally derived from the embryonalmesoderm and isolated from adult bone marrow, can differentiate to formmuscle, bone, cartilage, fat, marrow stroma, and tendon. Methods ofisolating and culturing MSC are known in the art; and any known methodcan be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, whichdescribes isolation and culture of human MSC.

Suitable parent cells can be euploid, e.g., have one of the followingkaryotypes: 46XY and 46XX. In some cases, parent cells are aneuploid.

Suitable parent cells include cells that are free of intracellularpathogens, e.g., do not have a detectable intracellular pathogen. Forexample, a suitable parent cell in which one or more intracellularpathogens cannot be detected. Example of such intracellular pathogensinclude, e.g., human papilloma virus (HPV), cytomegalovirus (CMV),Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpessimplex virus (HSV), mycoplasma, rickettsia, replication-competentretroviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus,and the like.

Suitable parent cells that do not include known deleterious mutations,where known deleterious mutations include, but are not limited to,deleterious mutations in a structural protein, and deleterious mutationsin a functional protein, e.g., an enzyme, a transcription factor, atumor suppressor; deleterious mutations in a non-coding region of agene; loss of a coding region for a protein; mutations resulting inabnormal methylation patterns; and the like. Examples of knowndeleterious mutations include, but are not limited to, a mutation incystic fibrosis transmembrane conductance regulator (CFTCR) that leadsto cystic fibrosis (e.g., a ΔF508 mutation; a G542X mutation; a G551Dmutation; an N1303K mutation; a W1282X; etc.); more than 40 CAG repeatsin the huntingtin gene; a mutation in the adenosine deaminase gene; amutation in XMR1, e.g., CGG repeats in the 5′ untranslated region of theFMR1 gene; mutations in the p53 tumor suppressor gene; mutations in Ras;mutations in type I collagen genes COL1A1 and COL1A2 that causeosteogenesis imperfecta; globlin mutations that case sickle cell anemiaor thalassemia; mutations in clotting factor genes that causehemophilia; mutations in dystrophin that cause muscular dystrophy; andthe like.

Suitable parent cells also include cells that do not have any grosschromosomal abnormalities that can be detected by karyotyping, e.g.,aneuploidy of any chromosome, e.g., chromosome 21 trisomy, chromosome 18trisomy; deletions of a segment of a chromosome; inversion of a segmentof a chromosome; duplication of a segment of a chromosome; loss of aportion of a chromatid arm; translocation of all or a part of achromosome to another chromosome; and the like.

Before genetically modifying a parent HLA heterozygous cell such that itis HLA homozygous, the parent cell can be manipulated in any of a numberof ways (e.g., by genetic modification, selection, etc.), as describedbelow.

An HLA heterozygous parent cell can be genetically modified before beinggenetically modified to become HLA homozygous. For example, an HLAheterozygous parent cell can be genetically modified with a nucleic acidthat comprises a nucleotide sequence encoding a therapeutic protein,such that the therapeutic protein is produced by the cell (e.g, in aconstitutive or an inducible manner). Non-limiting examples of suitabletherapeutic proteins include, but are not limited to, cytokines;lymphokines; erythropoietin; colony stimulating factors; growth factors;an interleukin; clotting factors; glucose level regulating proteins(e.g., insulin); growth hormones; tumoricidal proteins; anti-angiogenicproteins; and the like.

As another example, a deleterious mutation in a parent HLA heterozygouscell can be corrected before making the cell HLA homozygous. Any knownmethod can be used to correct a deleterious mutation. See, e.g., WO98/48005; Inoue et al. (2001) Mol. Ther. 3:526-530; and Miller et al.(2006) Nat. Biotech. 24:1022. Where an HLA heterozygous parent cell hasDNA encoding transposons or viral remnants in its nuclear genome, suchDNA can be removed. Furthermore, a parent cell can be geneticallymodified with one or more suicide genes that could be activated to as tokill a transplanted cell.

Genetically Modified MLA Homozygous Cells

A subject HLA homozygous cell is homozygous for an HLA haplotype that iscompatible with at least one prospective recipient. A subject HLAhomozygous cell can be homozygous for an HLA haplotype that is found infrom about 0.01% to about 25% or more of a selected human population.For example, a subject HLA homozygous cell can be homozygous for an HLAhaplotype found in from about 0.01% to about 25% or more of a Caucasianpopulation, an Asian population (e.g., Chinese, Japanese, Taiwanese,Korean, etc.), an African-American population, an Hispanic population,etc.

Currently known HLA class I and class II alleles are depicted in FIGS. 3and 4, respectively. The human HLA locus is depicted schematically inFIG. 5. An HLA haplotype is a collection of HLA class I and class IIalleles found on a given chromosome in an individual. A subject HLAhomozygous cell or progeny thereof, is homozygous for the entire HLAlocus, including class I and class II HLA molecule-encoding portions ofthe locus. HLA-A,B haplotype coverage in U.S. populations is depicted inFIG. 6. Common HLA-A,B haplotypes in five U.S. populations are depictedin FIG. 7, where the following abbreviations are used: AFAM, AfricanAmerican; CAU, Caucasian; ORI, Oriental (e.g., Asian): HIS, Hispanic;and NAI, Native American Indian. See, e.g., Cao et al. (2001) HumanImmunol. 62:1009. A subject HLA homozygous cell can include one of thecommon HLA-A,B haplotypes depicted in FIG. 7.

As described in more detail below, a subject HLA homozygous cell can begenerated in a process that involves genetic modification of a parentstem cell with a recombinant targeting parvoviral vector, followed bynegative selection to select for loss of the negative selection marker.Negative selection results in loss of the negative selection marker andthe entire recombinant parvoviral vector, such that a subject HLAhomozygous cell does not contain any detectable parvoviral sequencesfrom the recombinant parvoviral vector that integrated into the genomeduring the positive selection step.

As noted above, a parent cell that is HLA heterozygous (i.e.,heterozygous at the HLA locus) is genetically modified such that it ishomozygous at the HLA locus, generating a genetically modified HLAhomozygous cell. A subject HLA homozygous cell can be homozygous at oneor more loci other than the HLA locus; however, a majority of thenon-HLA loci in a subject HLA homozygous cells are heterozygous. Forexample, a subject HLA homozygous cell comprises a genome in which atleast about 50%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, or more (e.g., 99.5%, 99.9%, etc.), of the loci otherthan the HLA locus are heterozygous.

As noted above, a parent cell can be substantially free of one or moreintracellular pathogens, e.g., no detectable intracellular pathogen ispresent in a parent stem cell. Similarly, a subject HLA homozygous cellcan be substantially free of intracellular pathogens. Thus, e.g., asubject HLA homozygous cell can have no detectable intracellularpathogen, where intracellular pathogens include, e.g., HPC, EBV, CMV,HIV, HSV, HCV, HBV, HAV, mycoplasma, and the like. Whether a subject HLAhomozygous cell is free of one or more intracellular pathogens isreadily determined using any of a variety of methods, including, e.g.,polymerase chain reaction (PCR) analysis of genomic DNA using primersspecific for a particular pathogen (e.g., where pathogen nucleic acid isgenomically integrated); and the like.

A parent stem cell generally does not have any detectable grosschromosomal abnormalities, e.g., chromosomal abnormalities that can bedetected by karyotype. Similarly, subject HLA homozygous cell does nothave any detectable gross chromosomal abnormalities that can be detectedby karyotyping, e.g., aneuploidy of any chromosome, e.g., chromosome 21trisomy, chromosome 18 trisomy; deletions of a segment of a chromosome;inversion of a segment of a chromosome; duplication of a segment of achromosome; loss of a portion of a chromatid arm; translocation of allor a part of a chromosome to another chromosome; and the like. An HLAhomozygous cell can be euploid or aneuploid. In some cases, an HLAhomozygous cell is euploid.

Analysis of the nuclear chromosomal karyotype can be used to assessgenetic stability of an HLA homozygous cell or progeny thereof. Forexample, an HLA homozygous cell that is maintained in culture for anextended period of time can be periodically subjected to analysis of thenuclear chromosomal karyotype to determine whether any gross chromosomalabnormalities are present and detectable.

A parent cell can be one that lacks one or more known deleteriousmutations. Similarly, a subject HLA homozygous can lack one or moreknown deleterious mutations, where known deleterious mutations include,but are not limited to, deleterious mutations in a structural protein,and deleterious mutations in a functional protein, e.g., an enzyme, atranscription factor, a tumor suppressor; deleterious mutations in anon-coding region of a gene; loss of a coding region for a protein;mutations resulting in abnormal methylation patterns; and the like.

Whether a subject HLA homozygous cell or progeny thereof contains anydeleterious mutations can be readily determined using DNA microarrayanalysis, proteomic analysis, or other method for detecting the presenceof a mutation.

Furthermore, a subject HLA homozygous cell can be one that does notundergo uncontrolled cell proliferation, i.e., is not tumorigenic.

Further Genetic Modifications

A subject HLA homozygous cell can be further genetically modified,depending on various factors, including the therapeutic purpose forwhich the HLA homozygous cell is intended.

For example, an HLA homozygous cell can be genetically modified toinclude a nucleic acid comprising a nucleotide sequence encoding atherapeutic polypeptide, e.g., a polypeptide that is need by the hostand that the host does not produce; a polypeptide that the hostproduces, but in insufficient quantities; and a functional version of amutant, non-functional polypeptide that the host produces. Non-limitingexamples of such therapeutic proteins include, but are not limited to,cytokines; lymphokines; erythropoietin; colony stimulating factors;growth factors; an interleukin; clotting factors; glucose levelregulating proteins (e.g., insulin); growth hormones; and the like.

Selection for Differentiated Cell Types

Depending on the therapeutic application in which a subject HLAhomozygous cell is to be used, the HLA homozygous cell can bemanipulated in in vitro culture. For example, a subject HLA homozygousstem cell or progenitor cell can be induced in vitro to differentiateinto a desired cell type. Where a particular cell type is desired, anHLA homozygous stem cell is cultured under conditions that provide fordifferentiation into the cell type. Cells that have differentiated intoa desired cell type are readily identified by the presence of one ormore cell markers that are indicators of the cell type. The presentinvention provides differentiated HLA homozygous cells, e.g., HLAhomozygous cardiomyocytes, HLA homozygous neurons, HLA homozygous glialcells, HLA homozygous mesenchymal lineage cells, HLA homozygouspancreatic islet cells, etc.

Methods of inducing a stem cell to undergo differentiation in vitro areknown in the art; and any known method can be used. See, e.g., Odoricoet al. ((2001) Stem Cells 19:193-204).

A subject method can involve: a) inducing differentiation in apopulation of HLA homozygous stem cells, generating a mixed cellpopulation that includes undifferentiated stem cells and differentiatedcells; and b) separating the differentiated cells from theundifferentiated cells. As an example, a subject method can involve: a)inducing cardiomyogenesis in a population of HLA homozygous stem cells,generating a mixed population of undifferentiated stem cells andcardiomyocytes; and b) separating cardiomyocytes from theundifferentiated (non-cardiomyocyte) cells. The separation step caninvolve contacting the mixed cell population with an antibody specificfor a cardiomyocyte-specific cell surface marker.

Neuronal Cells and Glial Cells

For example, under certain in vitro culture conditions, a stem cell canbe induced to differentiate into a neuronal cell, an astrocyte, anoligodendrocyte, or a neuronal precursor cell. As an example, an HLAhomozygous cell can be cultured in the presence of ligands that bindgrowth factor receptors to promote enrichment for neural precursorcells. The growth environment may contain a neural cell supportiveextracellular matrix, such as fibronectin. Other methods for inducingdifferentiation of an ES cell into a neuronal precursor cell aredescribed in, e.g., U.S. Pat. No. 6,887,706; and U.S. Pat. No.7,011,828. Markers of interest include, but are not limited to,β-tubulin III or microtubule-associated protein 2 (MAP-2),characteristic of neurons; glial fibrillary acidic protein (GFAP),present in astrocytes; galactocerebroside (GalC) or myelin basic protein(MBP); characteristic of oligodendrocytes; Nestin or Musashi,characteristic of neural precursors and other cells. A mature neuronalcell can be characterized by an ability to express one, two, three,four, five, six, seven, or all eight of: 160 kDa neuro-filament protein,MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase (GAD),tyrosine hydroxylase, GABA, and serotonin. The differentiated cellsforming neural progenitor cells, neuron cells and/or glial cells canalso be characterized by expressed markers characteristic ofdifferentiating cells. The in vitro differentiated cell culture can beidentified by detecting molecules such as markers of the neuroectodermallineage, markers of neural progenitor cells, neuro-filament proteins,MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase, GABA,serotonin, tyrosine hydroxylase, β-tubulin, β-tubulin III, GABA Aα2receptor, glial fibrillary acidic protein (GFAP), 2′, 3′-cyclicnucleotide 3′-phosphodiesterase (CNPase), plp, DM-20, O4, and NG-2staining.

Hepatocytes

As another example, an HLA homozygous stem cell can be cultured in thepresence of a hepatocyte differentiation agent to promote enrichment forhepatocyte-like cells. The growth environment may contain a hepatocytesupportive extracellular matrix, such as collagen or Matrigel™. Suitabledifferentiation agents include various isomers of butyrate and theiranalogs, exemplified by n-butyrate. The cultured cells are optionallycultured simultaneously or sequentially with a hepatocyte maturationfactor, such as an organic solvent like dimethyl sulfoxide (DMSO); amaturation cofactor such as retinoic acid; or a cytokine or hormone suchas a glucocorticoid, epidermal growth factor (EGF), insulin,transforming growth factors (TGF-α and TGF-β), fibroblast growth factors(FGF), heparin, hepatocyte growth factors (HGF), interleukins (IL-1 andIL-6), insulin-like growth factors (IGF-I and IGF-II), andheparin-binding growth factors (HBGF-1). Hepatocyte lineage cellsdifferentiated from stem cells can display one, two, three, or more, ofthe following markers: α₁-antitrypsin (AAT) synthesis, albuminsynthesis, asialoglycoprotein receptor (ASGR) expression, absence ofα-fetoprotein, evidence of glycogen storage, evidence of cytochrome p450activity, and evidence of glucose-6-phosphatase activity.

Cardiomyocytes

An HLA homozygous stem cell can be induced in vitro to differentiateinto a cardiomyocyte, generating an HLA homozygous cardiomyocyte. An HLAhomozygous cardiomyocyte can be used to treat, e.g., areas of ischemiccardiac tissue resulting from myocardial infarction; heart failure; andthe like. Suitable cardiomyocyte-specific cell surface markers include,but are not limited to, troponin and tropomyosin.

Mesenchymal Lineages

An HLA homozygous stem cell can be induced in vitro to differentiateinto a cell of a mesenchymal lineage, e.g., a lineage selected fromosteogenic, chondrogenic, tendonogenic, ligamentogenic, myogenic, marrowstromagenic, adipogenic and dermogenic. Thus, e.g., an HLA homozygousmesenchymal stem cell can be induced in vitro to differentiate into anosteoblast, a chondrocyte, a myoblast, a stromal cell, etc., using anyknown method. See, e.g., U.S. Pat. No. 5,736,396, which describesmethods for in vitro differentiation of human mesenchymal stem cells.

Pancreatic Islet Cells

An HLA homozygous stem cell can be induced in vitro to differentiateinto a pancreatic islet cell, generating an HLA homozygous pancreaticislet cell, which can be used to treat Type 1 diabetes. Methods ofinducing differentiation into a pancreatic islet cell are described in,e.g., Zulewski (2006) Swiss Med. Weekly 136(41-42):647-54; Trounson(2006) Endocrin. Rev. 27(2):208-19; Soria et al. (2005) Novartis Found.Symp. 265:158-67; and Xu et al. (2006) Cloning and Stem Cells 8:96-107.

Separation of Differentiated Cells from Undifferentiated Cells

As noted above, where a population of HLA homozygous stem cells isinduced in vitro to differentiate, a mixed cell population can result,where the mixed cell population includes undifferentiated HLA homozygouscells and differentiated HLA homozygous cells. Where it is desired togenerate a population of HLA homozygous cells in which most orsubstantially all of the cells are HLA homozygous differentiated cells,the differentiated cells can be separated from the undifferentiatedcells. Separation can be carried out on the basis of cell surfacemarkers expressed by differentiated cells, but not by undifferentiatedcells. Suitable cell surface markers for differentiated cells of variouscell types are known in the art, and are described above.

Separation can be carried out using well-known methods, including, e.g.,any of a variety of sorting methods, e.g., fluorescence activated cellsorting (FACS), negative selection methods, etc. The selected(differentiated) cells are separated from non-selected(undifferentiated) cells, generating a population of selected (“sorted”)cells. A selected cell population can be at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or greater than 99%differentiated cells of a particular (selected) cell type.

Cell sorting (separation) methods are well known in the art. Proceduresfor separation may include magnetic separation, using antibody-coatedmagnetic beads, affinity chromatography and “panning” with antibodyattached to a solid matrix, e.g. plate, or other convenient technique.Techniques providing accurate separation include fluorescence activatedcell sorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. Dead cells may be eliminated byselection with dyes associated with dead cells (propidium iodide [PI],LDS). Any technique may be employed which is not unduly detrimental tothe viability of the selected cells. Where the selection involves use ofone or more antibodies, the antibodies can be conjugated with labels toallow for ease of separation of the particular cell type, e.g. magneticbeads; biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry.

Compositions Comprising HLA Homozygous Cells or Progeny

Also provided are compositions, including pharmaceutical compositions,comprising a subject HLA homozygous cell, or a progeny of such a cell,including, e.g., a differentiated progeny of an HLA homozygous stem cellor progenitor cell. A subject cell composition comprises an HLAhomozygous cell (e.g., an HLA homozygous stem cell, or a progenythereof) and one or more additional components. Suitable componentsinclude, but are not limited to, salts; buffers; stabilizers;protease-inhibiting agents; cell membrane- and/or cell wall-preservingcompounds, e.g., glycerol, dimethylsulfoxide, etc.; nutritional mediaappropriate to the cell; and the like.

For administration to a mammalian host, a subject HLA homozygous cellcan be formulated as a pharmaceutical composition, where apharmaceutical composition comprises, in addition to a subjecthomozygous cell, at least one pharmaceutically acceptable excipient.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy,” 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

A pharmaceutical composition can be a sterile aqueous or non-aqueoussolution, suspension or emulsion, which additionally comprises aphysiologically acceptable carrier (i.e., a non-toxic material that doesnot substantially adversely affect cell viability or activity). Anysuitable carrier known to those of ordinary skill in the art may beemployed in a subject pharmaceutical composition. The selection of acarrier will depend, in part, on the nature of the cells beingadministered. Representative carriers include physiological salinesolutions, gelatin, water, alcohols, natural or synthetic oils,saccharide solutions, glycols, injectable organic esters such as ethyloleate or a combination of such materials. Optionally, a pharmaceuticalcomposition can additionally contain preservatives and/or otheradditives such as, for example, antimicrobial agents, anti-oxidants,chelating agents and/or inert gases, and/or other active ingredients.

A subject HLA homozygous cell or progeny thereof can be encapsulated,according to known encapsulation technologies, includingmicroencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and5,084,350). Where a subject HLA homozygous stem cell or progeny thereofis encapsulated, the HLA homozygous stem cell or progeny thereof can beencapsulated by macroencapsulation, as described in U.S. Pat. Nos.5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and published PCTpatent application WO 95/05452.

A subject HLA homozygous stem cell or progeny thereof can be present ina matrix. The term “matrix” refers to any suitable carrier material towhich the cells are able to attach themselves or adhere in order to forma corresponding cell composite, e.g., artificial tissue. In someembodiments, the matrix or carrier material, respectively, is presentalready in a three-dimensional form desired for later application. Forexample, bovine pericardial tissue is used as matrix which iscrosslinked with collagen, decellularized and photofixed.

As an example, a matrix (also referred to as a “biocompatiblesubstrate”) is a material that is suitable for implantation into asubject onto which a cell population can be deposited. A biocompatiblesubstrate does not cause toxic or injurious effects once implanted inthe subject. In one embodiment, the biocompatible substrate is a polymerwith a surface that can be shaped into the desired structure thatrequires repairing or replacing. The polymer can also be shaped into apart of a structure that requires repairing or replacing. Thebiocompatible substrate provides the supportive framework that allowscells to attach to it, and grow on it. Cultured populations of cells canthen be grown on the biocompatible substrate, which provides theappropriate interstitial distances required for cell-cell interaction.

A subject HLA homozygous cell or progeny thereof can be cryopreservedaccording to routine procedures. For example, cryopreservation can becarried out on from about one to ten million cells in “freeze” mediumwhich can include a suitable proliferation medium, 10% serum albumin(e.g., human serum albumin) and 7.5% dimethylsulfoxide. Cells arecentrifuged. Growth medium is aspirated and replaced with freeze medium.Cells are resuspended as spheres. Cells are slowly frozen, by, e.g.,placing in a container at −80° C. Cells are thawed by swirling in a 37°C. bath, resuspended in fresh culture medium.

Where desired, the differentiated cells can be formed into a tissuesheet, for use in transplantation into a recipient. A tissue sheetcomprising, e.g., living HLA homozygous, differentiated cells andextracellular matrix is formed using any known method. See, e.g., U.S.Pat. No. 7,166,464.

HLA Homozygous Cell Library

As discussed above, a library of HLA homozygous cells is provided. Alibrary includes one, two, three, four, five, six, seven, eight, nine,10, 10-15, 15-20, 20-25, 25-30, or more than 30 different types of HLAhomozygous cells. A subject library can include a first HLA homozygouscell homozygous for a first HLA haplotype; and at least a second HLAhomozygous cell homozygous for a second HLA haplotype.

For example, a subject library can include a first HLA homozygous cellhomozygous for a first HLA haplotype; a second HLA homozygous cellhomozygous for a second HLA haplotype; a third HLA homozygous cellhomozygous for a third HLA haplotype; a fourth HLA homozygous cellhomozygous for a fourth HLA haplotype; and so on.

The different HLA haplotypes represented in a subject library canrepresent major HLA haplotypes found in various human populations, e.g.,one or more major HLA haplotypes found in a Caucasian population; one ormore major HLA haplotypes found in an African-American population; oneor more major HLA haplotypes found in a Chinese population; one or moremajor HLA haplotypes found in a Japanese population; one or more majorHLA haplotypes found in an Hispanic population; and the like.

A subject library can include a single cell type or can include two ormore different cell types.

For example, a subject library can be a library of HLA homozygous cells,where all of the cells are of the same cell type. For example, a subjectlibrary can include a first HLA homozygous cardiomyocyte homozygous fora first HLA haplotype; a second HLA homozygous cardiomyocyte homozygousfor a second HLA haplotype; a third HLA homozygous cardiomyocytehomozygous for a third HLA haplotype; a fourth HLA homozygouscardiomyocyte homozygous for a fourth HLA haplotype; and so on.

Alternatively, a subject library can include two or more different celltypes. For example, the library can include a pancreatic islet cell, acardiomyocyte, a keratinocyte, etc., where all of the cells arehomozygous for the same HLA haplotype. In some cases, a subject librarywill include two or more sub-libraries, e.g., the library can include asub-library of HLA homozygous pancreatic islet cells, including, e.g., afirst HLA homozygous pancreatic islet cell of a first HLA haplotype, asecond HLA homozygous pancreatic islet cell of a second HLA haplotype,etc.; a sub-library of HLA homozygous cardiomyocytes, including, e.g., afirst HLA homozygous cardiomyocyte of a first HLA haplotype, a secondHLA homozygous cardiomyocyte of a second HLA haplotype, etc.; asub-library of keratinocytes; and so on.

In some embodiments, a subject library is a library of HLA homozygousstem cells. For example, a subject library can include a first HLAhomozygous stem cell homozygous for a first HLA haplotype; a second HLAhomozygous stem cell homozygous for a second HLA haplotype; a third HLAhomozygous stem cell homozygous for a third HLA haplotype; a fourth HLAhomozygous stem cell homozygous for a fourth HLA haplotype; and so on.

A subject library can be catalogued, e.g., by a searchable computerdatabase, in which information regarding the HLA haplotype, andoptionally additional information such as cell surface markers,karyotype information, and the like, is stored and can be searched.

Methods of Generating an HLA Homozygous Cell

The present invention further provides methods of generating an HLAhomozygous cell or progeny thereof, including differentiated progeny.The methods generally involve genetically modifying an HLA heterozygousparent cell with a targeting vector comprising: a) a targetingnucleotide sequence that provides for homologous recombination with atarget nucleotide sequence centromeric to a HLA locus on a first copy ofchromosome 6 in the parent stem cell; b) a selectable marker(s) thatprovides for selection for integration of the targeting nucleotidesequence into the chromosome, and for selection for homozygosity at theHLA locus, where the selectable marker can be located within thetargeting nucleotide sequence, to generate a genetically modified parentcell that includes the targeting sequence integrated into a first copyof chromosome 6 at a site centromeric to the HLA locus.

Selection of the genetically modified cell is then carried out. Theabove-described genetic modification can result in a mixed population ofcells, where a proportion of the cells include the targeting sequenceintegrated into a first copy of chromosome 6 at a site centromeric tothe HLA locus. Selection of cells that include the targeting sequenceintegrated into a first copy of chromosome 6 at a site centromeric tothe HLA locus is carried out. Genetically modified parent cells thatinclude the targeting sequence integrated into a first copy ofchromosome 6 at a site centromeric to the HLA locus are then selectedfor duplication of one copy of the HLA locus in such a manner that bothcopies of chromosome 6 have the same HLA haplotype.

Any of a number of targeting vectors can be used. Parvoviral vectors areexemplified below. However, the method is not limited to use of aparvoviral targeting vector.

As noted above, a subject method can be carried out using a parvoviraltargeting vector. In this case, the methods generally involvegenetically modifying an HLA heterozygous parent cell with a parvoviralvector comprising: a) a targeting nucleotide sequence that provides forhomologous recombination with a target nucleotide sequence centromericto a HLA locus on a first copy of chromosome 6 in the parent cell; b) aselectable marker(s) that provides for selection for integration of thetargeting nucleotide sequence into the chromosome, and for selection forhomozygosity at the HLA locus, where the selectable marker(s) is locatedwithin the targeting nucleotide sequence; and c) at least one invertedterminal repeat, or a functional equivalent thereof, flanking thetargeting nucleotide sequence, to generate a genetically modified parentcell. Selection steps are carried out as described above.

In some cases, both positive and negative selection in used. In thesecases, the methods involve genetically modifying an HLA heterozygousparent cell with a parvoviral vector comprising: a) a targetingnucleotide sequence that provides for homologous recombination with atarget nucleotide sequence centromeric to a HLA locus on a first copy ofchromosome 6 in the parent cell; b) a selectable marker(s) that providesfor positive and negative selection, where the selectable marker islocated within the targeting nucleotide sequence; and c) at least oneinverted terminal repeat, or a functional equivalent thereof, flankingthe targeting nucleotide sequence, to generate a genetically modifiedparent cell.

The genetically modified stem cell is cultured under conditions thatprovide for positive selection and negative selection of the selectablemarker. Positive selection results in a genetically modified parent stemcell comprising the parvoviral vector integrated into the targetnucleotide sequence on the first copy of chromosome 6 at a locationcentromeric to the HLA locus. Negative selection results in agenetically modified stem cell that is homozygous for the HLA haplotypepresent on the second copy of chromosome 6. Negative selection alsoresults in loss of the HLA locus and the parvoviral vector present onthe first copy of chromosome 6.

Positive selection is carried out first, generating a geneticallymodified parent stem cell comprising the parvoviral vector integratedinto the target nucleotide sequence on the first copy of chromosome 6 ata location centromeric to the HLA locus. Negative selection can becarried out following positive selection, e.g., negative selection canbe carried out directly following the completion of positive selection.Alternatively, the genetically modified parent cell comprising therecombinant parvoviral vector integrated into the target nucleotidesequence on the first copy of chromosome 6 at a location centromeric tothe HLA locus can be stored (e.g., cryopreserved); and negativeselection can be performed at a later time (e.g., one day, two days toone week, one week to one month, one month to six months, or six monthsto one year, or later, following positive selection).

Modification of Other Loci

Described above are methods of generating a cell that is homozygous atthe HLA locus. While the methods focus on the HLA locus, the same methodcan be applied to other loci, e.g., to generate a cell that ishomozygous at a locus of interest. The present invention thus providesmethods of generating a cell that is homozygous at a locus of interest;as well as cells generated by the method. The methods generally involvegenetically modifying a parent cell that is heterozygous at a locus ofinterest with a targeting vector comprising: a) a targeting nucleotidesequence that provides for homologous recombination with a targetnucleotide sequence centromeric to the locus of interest on a first copyof a chromosome in the parent stem cell; b) a selectable marker(s) thatprovides for selection for integration of the targeting nucleotidesequence into the first copy of the chromosome, where the selectablemarker can be located within the targeting nucleotide sequence, togenerate a genetically modified parent cell that includes the targetingsequence integrated into a first copy of the chromosome at a sitecentromeric to the locus of interest. Selection steps are carried out asdescribed above, to generate a genetically modified cell that ishomozygous at the locus of interest.

Recombinant Parvoviral Vector and Virion

The present invention also provides a recombinant targeting parvoviralvector (e.g., a recombinant targeting MVM vector; a recombinanttargeting AAV vector) comprising: a) a targeting nucleotide sequencethat provides for homologous recombination with a target nucleotidesequence centromeric to a histocompatibility locus; b) a selectablemarker that provides for positive and negative selection, wherein theselectable marker is located within the targeting nucleotide sequence;and c) at least one inverted terminal repeat, or a functional equivalentthereof, flanking the targeting nucleotide sequence. The presentinvention also provides rAAV virions generated using a subject rAAV,where the rAAV virions are suitable for infecting a parent, HLAheterozygous, stem cell.

As noted above, the recombinant parvoviral vector that is used togenerate a subject HLA homozygous stem cell includes: a) a targetingnucleotide sequence that provides for homologous recombination with atarget nucleotide sequence centromeric to a HLA locus on a first copy ofchromosome 6 in a parent stem cell; b) a selectable marker(s) thatprovides for positive and negative selection, where the selectablemarker is located within the targeting nucleotide sequence; and c) atleast one inverted terminal repeat, or a functional equivalent thereof,flanking the targeting nucleotide sequence.

The target nucleotide sequence can be any sequence present between theHLA locus and the centromere. As one non-limiting example, the targetnucleotide sequence is a nucleotide sequence present in the HMGA1 gene.For example, the target nucleotide sequence can include HMGA1 exon 3nucleotide sequences.

Selectable markers suitable for positive selection include, but are notlimited to, nucleic acids comprising nucleotide sequences encoding apolypeptide that provides resistance to antimicrobial agents. Suitableselectable markers that provide for positive selection include, but arenot limited to, a hygromycin B resistance gene (encoding aminoglycosidephosphotranferase (APH)) that confers resistance to hygromycin; aneomycin phosphotranferase gene (encoding neomycin phosphotransferase)that confers resistance to G418; pac, which confers resistance topuromycin; a zeomycin resistance gene, which confers resistance tozeomycin; a BlaS gene conferring blasticidin resistance; and the like.

Selectable markers suitable for negative selection include, but are notlimited to, nucleic acids comprising nucleotide sequences encodingthymidine kinase, which confers sensitivity to gancyclovir.

A nucleic acid comprising a nucleotide sequence encoding a fusionprotein that provides for both positive and negative can also beincluded in the parvoviral vector. For example, a fusion polypeptidecomprising a hygromycin resistance portion and a thymidine kinaseportion can be used. See, e.g., Lupton et al. (1991) Mol. Cell Biol.11:3374-3378. As another example, a fusion polypeptide comprising ablasticidin resistance portion and a thymidine kinase portion can beused. See, e.g., Karreman (1998) Nucl. Acids Res. 26:2508-2510.

As noted above, the selectable marker(s) are located within thetargeting sequence. The total combined length of the targeting sequenceand selectable marker(s) is generally less than about 5 kilobases (kb).

The parvoviral vector includes at least one inverted terminal repeat, ora functional equivalent thereof, flanking the targeting nucleotidesequence. The nucleotide sequences of AAV UR regions are known. See,e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence.AAV ITRs used in the vectors of the invention need not have a wild-typenucleotide sequence, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides. Additionally, AAV ITRs may bederived from any of several AAV serotypes, including without limitation,AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5′ and 3′ITRs which flank a selected nucleotide sequence in an AAV expressionvector need not necessarily be identical or derived from the same AAVserotype or isolate, so long as they function as intended, i.e., toallow for excision and rescue of the sequence of interest from a hostcell genome or vector, and to allow integration of the DNA molecule intothe recipient cell genome when AAV Rep gene products are present in thecell. ITRs allow replication of the vector sequence in the presence ofan appropriate mixture of Rep proteins. ITRs also allow for theincorporation of the vector sequence into the capsid to generate an AAVparticle.

Genetically Modified Parent Cells Comprising Integrated RecombinantParvoviral Vector

As noted above, positive selection of a genetically modified parent cellresults in a genetically modified parent cell comprising the targetingvector integrated into the target nucleotide sequence on the first copyof chromosome 6 at a location centromeric to the HLA locus. The presentinvention also provides a genetically modified parent cell comprisingthe targeting vector integrated into the target nucleotide sequence onthe first copy of chromosome 6 at a location centromeric to the HLAlocus. The genetically modified parent cell comprising the targetingvector integrated into the target nucleotide sequence on the first copyof chromosome 6 at a location centromeric to the HLA locus can be stored(e.g., cryopreserved) and kept for days, weeks, months, or years.

Generating an rAAV Virion

As noted above, the recombinant parvoviral vector can be a recombinantAAV (rAAV) vector. Methods of generating a recombinant AAV (rAAV) vectorfor use in a subject method are described in, e.g., Hirata and Russell(2000) J. Virol. 74:4612-4620. By way of introduction, it is typical toemploy a host or “producer” cell for rAAV vector replication andpackaging. Such a producer cell (usually a mammalian host cell)generally comprises or is modified to comprise several different typesof components for rAAV production. The first component is a recombinantadeno-associated viral (rAAV) vector genome (or “rAAV pro-vector”) thatcan be replicated and packaged into vector particles by the hostpackaging cell. The rAAV pro-vector will normally comprise aheterologous polynucleotide (or “transgene”), with which it is desiredto genetically alter another cell. The transgene is generally flanked byone or two AAV inverted terminal repeats (ITRs) which comprise sequencesthat are recognized during excision, replication and packaging of theAAV vector, as well as during integration of the vector into a host cellgenome.

A second component is a helper virus that can provide helper functionsfor AAV replication. Although adenovirus is commonly employed, otherhelper viruses can also be used as is known in the art. Alternatively,the requisite helper virus functions can be isolated genetically from ahelper virus and the encoding genes can be used to provide helper virusfunctions in trans. The AAV vector elements and the helper virus (orhelper virus functions) can be introduced into the host cell eithersimultaneously or sequentially in any order.

The final components for AAV production to be provided in the producercell are “AAV packaging genes” such as AAV rep and cap genes thatprovide replication and encapsidation proteins, respectively. Severaldifferent versions of AAV packaging genes can be provided (includingrep-cap cassettes and separate rep and/or cap cassettes in which the repand/or cap genes can be left under the control of the native promotersor operably linked to heterologous promoters. Such AAV packaging genescan be introduced either transiently or stably into the host packagingcell, as is known in the art and described in more detail below.

1. rAAV Vector

A subject rAAV virion, including the targeting sequences and selectablemarker(s), can be produced using standard methodology, known to those ofskill in the art. The methods generally involve: (1) introducing asubject rAAV vector into a host cell; (2) introducing an AAV helperconstruct into the host cell, where the helper construct includes AAVcoding regions capable of being expressed in the host cell to complementAAV helper functions missing from the AAV vector, (3) introducing one ormore helper viruses and/or accessory function vectors into the hostcell, wherein the helper virus and/or accessory function vectors provideaccessory functions capable of supporting efficient recombinant AAV(“rAAV”) virion production in the host cell; and (4) culturing the hostcell to produce rAAV virions. The rAAV vector, AAV helper construct andthe helper virus or accessory function vector(s) can be introduced intothe host cell, either simultaneously or serially, using standardtransfection techniques.

The rAAV vector, can be constructed by directly inserting the targetingsequences and the selectable marker sequences into an AAV genome whichhas had the major AAV open reading frames (“ORFs”) excised therefrom.Other portions of the AAV genome can also be deleted, so long as asufficient portion of the ITRs remain to allow for replication andpackaging functions. Such constructs can be designed using techniqueswell known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and5,139,941; International Publication Nos. WO 92/01070 (published Jan.23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al.(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90(Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) CurrentOpinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topicsin Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human GeneTherapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; andZhou et al. (1994) J. Exp. Med. 179:1867-1875.

2. AAV Helper Functions

Host cells containing the above-described rAAV vectors must be renderedcapable of providing AAV helper functions in order to replicate andencapsidate the nucleotide sequences flanked by the AAV ITRs to producerAAV virions. AAV helper functions are generally AAV-derived codingsequences which can be expressed to provide AAV gene products that, inturn, function in trans for productive AAV replication. AAV helperfunctions are used herein to complement necessary AAV functions that aremissing from the rAAV vectors. Thus, AAV helper functions include one,or both of the major AAV ORFs, namely the rep and cap coding regions, orfunctional homologues thereof. In the context of the instant invention,the cap functions include one or more mutant capsid proteins, wherein atleast one capsid protein comprises at least one mutation, as describedabove.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

AAV cap proteins include VP1, VP2, and VP3, wherein at least one of VP1,VP2, and VP3 comprises at least one mutation, as described above.

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the rAAV vector. AAV helperconstructs are thus used to provide at least transient expression of AAVrep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

Both rAAV vectors and AAV helper constructs can be constructed tocontain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to hygromycin; the neomycinphosphotranferase gene (encoding neomycin phosphotransferase) thatallows selection in mammalian cells by conferring resistance to G418;and the like. Other suitable markers are known to those of skill in theart.

3. AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding non AAV derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are non AAV derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

Particularly, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon,cosmid, or another virus. Accessory vectors can also be in the form ofone or more linearized DNA or RNA fragments which, when associated withthe appropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions.

Nucleic acid sequences providing the accessory functions can be obtainedfrom natural sources, such as from the genome of an adenovirus particle,or constructed using recombinant or synthetic methods known in the art.In this regard, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized. See, e.g.,Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRCHandbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N.(1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically,early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1bare thought to participate in the accessory process. Janik et al. (1981)Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessoryfunctions have been described. See, e.g., Young et al. (1979) Prog. Med.Virol. 25:113. Vaccinia virus-derived accessory functions have also beendescribed. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al.(1986) Virology 152:110-117.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest,e.g., the heterologous nucleic acid) from the rAAV vector. The Repproteins also serve to duplicate the AAV genome. The expressed Capproteins assemble into capsids, and the recombinant AAV genome ispackaged into the capsids. Thus, productive AAV replication ensues, andthe DNA is packaged into rAAV virions.

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas CsCl gradients. Further, if infection is employed to express theaccessory functions, residual helper virus can be inactivated, usingknown methods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions are then ready for use for geneticallymodifying a parent, HLA heterozygous, cell.

Therapeutic Methods

As noted above, a subject HLA homozygous cell or progeny thereof issuitable for use in a wide variety of clinical applications. As such,the present invention provides methods of treating various disorders,generally involving administering to an individual in need thereof aneffective number of subject HLA homozygous cells, or progeny thereof.

A subject HLA homozygous cell or progeny thereof is introduced into anindividual at a site that is appropriate to the disorder being treated.Sites and modes of administration can include, e.g., implantation (e.g.,of HLA homozygous cardiomyocytes) into heart muscle; intravenousinfusion (e.g., of HLA homozygous HSCs or HSC lineage cells);implantation into the pancreas (e.g., of HLA homozygous pancreatic isletcells); intracranial implantation (e.g., of HLA homozygous neural cellsor glial cells); and the like.

As noted above, the prospective recipient of a subject HLA homozygouscell is an individual who has two copies of an HLA locus and thereforetwo HLA haplotypes, where one HLA haplotype shares a substantialproportion of the alleles in the HLA haplotype of the HLA homozygouscell. A subject HLA homozygous cell is “HLA compatible” with aprospective recipient. “HLA compatible” means that some or all of theHLA alleles in the HLA haplotype of a subject HLA homozygous cell areshared by an HLA haplotype in a prospective recipient.

The individual can have an HLA haplotype that is identical to the HLAhaplotype of the HLA homozygous cell; however, the individual need nothave an HLA haplotype that is identical to the HLA haplotype of the HLAhomozygous cell. For example, the individual can have an HLA haplotypewhich shares from about 30% to about 40%, from about 40% to about 50%,from about 50% to about 60%, from about 60% to about 70%, from about 70%to about 80%, or from about 80% to about 90% of the alleles present inthe HLA haplotype of the HLA homozygous cell. In some instances, theindividual has an HLA haplotype having A and B alleles that areidentical to the HLA haplotype of the HLA homozygous cell. In somecases, the individual has an HLA haplotype having A, B, and DR allelesthat are identical to the HLA haplotype of the HLA homozygous cell. Insome cases, the individual has an HLA haplotype having A, B, Cw, and DRalleles that are identical to the HLA haplotype of the HLA homozygouscell. In some cases, the individual has an HLA haplotype having A, B,Cw, DR, and DQ alleles that are identical to the HLA haplotype of theHLA homozygous cell.

Where the individual has an HLA haplotype that is not identical to theHLA haplotype of the HLA homozygous cell, the individual can be treatedwith one or more immunosuppressive agents. However, the amount ofimmunosuppressive agent that is required to suppress rejection of thetransplant is less than the amount of immunosuppressive agent that isrequired to suppress rejection of a transplant that is less well matchedto the individual than a subject HLA homozygous cell.

An “effective amount” or “effective number” of HLA homozygous cells orprogeny is an amount or number that, when administered to an individualin one or more doses, provides a therapeutic effect. An effective numberof HLA homozygous cells or progeny thereof ranges from about 10³ cellsto about 10⁹ cells, e.g., from about 10³ cells to about 10⁴ cells, fromabout 10⁴ cells to about 10⁵ cells, from about 10⁵ cells to about 10⁶cells, from about 10⁶ cells to about 10⁷ cells, from about 10⁷ cells toabout 10⁸ cells, or from about 10⁸ cells to about 10⁹ cells.

As an example, an effective number of HLA homozygous pancreatic isletcells is a number of such cells that are effective to reduce a bloodglucose level in an individual by at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, or at least about 50% when compared to the bloodglucose levels in the absence of the cells. In some embodiments,effective number of HLA homozygous pancreatic islet cells is a numberthat is effective to reduce blood glucose levels to a normal range.Normal blood glucose levels are typically in the range of from about 70mg/dL to about 110 mg/dL before a meal (e.g., a fasting blood glucoselevel); and less than 120 mg/dL 2 hours after a meal.

As another example, an effective number of HLA homozygous cardiomocytesis a number of such cells that are effective to at least partiallyrestore cardiac function and/or to provide for an at least 10%, at least25%, at least 50%, or more, improvement in at least one parameter ofcardiac function. Cardiac function can be measured using standardmethods known to those skilled in the art.

A subject HLA homozygous cell, or an undifferentiated or differentiatedprogeny thereof is administered in a manner that permits the cell tograft to the intended tissue site and reconstitute or regenerate thefunctionally deficient area. The HLA homozygous stem cell or progenythereof used to treat a given disorder will depend in part on thediseased tissue to which function is to be restored. The following arenon-limiting examples.

Where a subject HLA homozygous cell is an HLA homozygous HSC, the HLAhomozygous HSC, or undifferentiated or differentiated progeny thereof,can be used in bone marrow transplantation to treat disorders such ascancer (e.g., leukemia); acquired immunodeficiency syndrome; sickle cellanemia; and various immune system disorders. An HLA homozygous HSC, orundifferentiated or differentiated progeny thereof, can be used in bonemarrow transplantation to treat an individual who has undergonechemotherapeutic treatment for a cancer and who is immunocompromised asa result.

A subject HLA homozygous cell can be induced to differentiate in vitrointo a cardiomyocyte, generating an HLA homozygous cardiomyocyte. An HLAhomozygous cardiomyocyte is used to treat cardiac diseases including,but not limited to, myocarditis, cardiomyopathy, heart failure, damagecaused by heart attacks (e.g., areas of ischemic heart tissue),hypertension, atherosclerosis, and heart valve dysfunction.

A subject HLA homozygous cell can be induced to differentiate in vitrointo a neuronal cell, or a glial cell, generating an HLA homozygousneuronal cell or glial cell. An HLA homozygous neuronal cell or glialcell can be used to treat a central nervous system (CNS) disorder. Forexample, an HLA homozygous neuronal cell or glial cell can be used totreat Alzheimer's disease, Parkinson's disease, Huntington's disease,AIDS associated dementia, spinal cord injury, and the like.

A subject HLA homozygous cell can be induced to differentiate in vitrointo a pancreatic islet cell, generating an HLA homozygous pancreaticislet cell. An HLA homozygous pancreatic islet cell can be used to treatType 1 diabetes.

A subject HLA homozygous cell (e.g., an HLA homozygous MSC) can beinduced to differentiate in vitro into a cartilage cell, generating anHLA homozygous cartilage cell. An HLA homozygous cartilage cell can beused to treat a disease of the joints or cartilage including but notlimited to cartilage tears, cartilage thinning, osteoporosis, andosteoarthritis.

A subject HLA homozygous cell can be induced to differentiate in vitrointo a renal cell, generating an HLA homozygous renal cell, which can beused to treat renal failure.

Subjects Suitable for Treatment

Subjects suitable for treatment with a subject method includeindividuals who have been diagnosed as having a blood cell cancer (e.g.,a leukemia); individuals who have been diagnosed with AIDS; individualswith sickle cell anemia; individuals with an immune disorder, e.g., anacquired immunodeficiency, a genetic immunodeficiency; individuals withType 1 diabetes; individuals with a nervous system disorder such asAlzheimer's disease, Parkinson's disease, spinal cord injury, stroke,etc.; individuals with a liver disorder such as hepatitis, cirrhosis, ametabolic disorder affecting the liver (e.g., lysosomal storagedisease); individuals with a disorder of the cartilage or bone, e.g.,individuals requiring joint replacement, individuals withosteoarthritis, individuals with osteoporosis, etc.; individuals with acardiac disorder, e.g., myocardial infarction, coronary artery disease,or other disorder resulting in ischemic cardiac tissue; individuals withrenal disorders, e.g., kidney failure (e.g., individuals on kidneydialysis); individuals with skeletal muscle disorders, such as musculardystrophy; and individuals with a lung disorder such as emphysema,pulmonary fibrosis, idiopathic pulmonary fibrosis, etc.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Generation of Human Stem Cells Homozygous at the HLA Locus

This Example describes generation of cell lines that have only oneversion of the HLA locus, instead of the two versions normally present(one on each copy of chromosome 6). The cell lines generated are thushomozygous at the HLA locus. Adeno-associated virus (AAV) vectors wereused to target genes near the HLA locus in stem cells and thereby tagthe locus for subsequent manipulations. The HMGA1 gene was targetedinitially, as it lies just centromeric to the HLA locus on chromosome 6.The targeting vector AAV2-HMGA12-HyTK is shown schematically in FIG. 1.This targeting vector is designed to introduce a HyTK gene into exon 3of the HMGA1 locus. The HyTK gene encodes a fusion protein with ahygromycin-resistance portion (for positive selection) and a thymidinekinase portion (for negative selection).

In the first step, H1 human embryonic stem cells were infected with theAAV2-HMGA1-HyTK vector, and several hygromycin-resistant colonies wereisolated. Based on Southern blot analysis, 4 of 4 lines analyzedcontained a single targeted locus.

In the second step, the lines generated in the first step were selectedfor loss of the HyTK transgene. Loss of the HyTK transgene could occurby homologous recombination between the 2 copies of chromosome 6, with acrossover occurring in a region centromeric to the HyTK transgene,resulting in a duplication of everything distal to the crossover. Such arecombination event should produce a cell with 2 copies of the HLA locusoriginally present on the untargeted chromosome, as depictedschematically in FIG. 2. Cells that were targeted at the HMGA1 locuswere selected with gancyclovir for loss of the HyTK transgene. TK⁻ cellsare gancyclovir resistant. One of three colonies analyzed had lost theHyTK transgene based on Southern blot analysis.

The Parental H1 human embryonic stem cells and the gancyclovir-resistantclone that lost the HyTK transgene were HLA typed. The original H1 linehad two different sets of HLA alleles: A02,03; B08,35; Cw07,04;DRB03,01; and DQ02,05. The gancyclovir-resistant clone had only one setof HLA alleles (i.e., one HLA haplotype), presumably in duplicate: A02;B08; Cw07; DRB03; DQ02. This cell line would therefore be ahistocompatible donor cell for any recipient with one copy of theA02-B08-Cw07-DRB03-DQ02 haplotype.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An isolated human stem cell or a progeny thereof, wherein the stemcell or progeny thereof is modified to be homozygous for a humanleukocyte antigen (HLA) haplotype, the HLA haplotype comprising anentire HLA locus; and wherein the cell is not homozygous at all loci. 2.The isolated human stem cell or progeny thereof of claim 1, wherein saidcell is selected from an embryonic stem cell, a neural stem cell, amesenchymal stem cell, a hematopoietic stem cell, and a differentiatedcell.
 3. The isolated human stem cell or progeny thereof of claim 1,wherein at least about 80% of the non-HLA loci in the cell areheterozygous.
 4. The isolated human stem cell or progeny thereof ofclaim 1, wherein at least about 90% of the non-HLA loci in the cell areheterozygous.
 5. The isolated human stem cell or progeny thereof ofclaim 1, wherein the HLA haplotype comprises alleles found in a commonHLA haplotype present in a selected human population.
 6. Adifferentiated progeny cell of the isolated human stem cell of claim 1,wherein the cell is selected from a neural cell, a glial cell, apancreatic islet cell, a cardiomyocyte, a mesenchymal lineage cell, anda hematopoietic stem cell lineage cell.
 7. A composition comprising theisolated human stem cell or progeny thereof of claim
 1. 8. A compositioncomprising the differentiated progeny cell of claim
 6. 9. Thecomposition of claim 8, wherein the differentiated progeny cell isassociated with a matrix.
 10. The composition of claim 8, wherein thedifferentiated progeny cell is present in a tissue sheet.